CN117607315A - High performance liquid chromatography for separating and detecting magnesium isoglycyrrhetate and related substances and application thereof - Google Patents
High performance liquid chromatography for separating and detecting magnesium isoglycyrrhetate and related substances and application thereof Download PDFInfo
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- CN117607315A CN117607315A CN202311586218.5A CN202311586218A CN117607315A CN 117607315 A CN117607315 A CN 117607315A CN 202311586218 A CN202311586218 A CN 202311586218A CN 117607315 A CN117607315 A CN 117607315A
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- 239000000126 substance Substances 0.000 title claims abstract description 154
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 title claims abstract description 64
- 229910052749 magnesium Inorganic materials 0.000 title claims abstract description 64
- 239000011777 magnesium Substances 0.000 title claims abstract description 64
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 29
- 238000001514 detection method Methods 0.000 claims abstract description 33
- 238000000926 separation method Methods 0.000 claims abstract description 31
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical group [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims abstract description 30
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 26
- WEVYAHXRMPXWCK-UHFFFAOYSA-N acetonitrile Substances CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 26
- 239000003814 drug Substances 0.000 claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 11
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical group CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 11
- 229940079593 drug Drugs 0.000 claims abstract description 10
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 47
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 34
- 239000007788 liquid Substances 0.000 claims description 21
- 239000011259 mixed solution Substances 0.000 claims description 21
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 17
- 238000002347 injection Methods 0.000 claims description 10
- 239000007924 injection Substances 0.000 claims description 10
- 239000002904 solvent Substances 0.000 claims description 9
- 238000007865 diluting Methods 0.000 claims description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000003643 water by type Substances 0.000 claims description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 2
- 241001251200 Agelas Species 0.000 claims description 2
- 238000010828 elution Methods 0.000 claims description 2
- 235000019253 formic acid Nutrition 0.000 claims description 2
- 239000003002 pH adjusting agent Substances 0.000 claims description 2
- 238000001228 spectrum Methods 0.000 claims description 2
- 239000012085 test solution Substances 0.000 claims description 2
- 238000004587 chromatography analysis Methods 0.000 claims 1
- 238000009472 formulation Methods 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 description 9
- 239000012535 impurity Substances 0.000 description 8
- 230000008901 benefit Effects 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 206010067484 Adverse reaction Diseases 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000006838 adverse reaction Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 208000008964 Chemical and Drug Induced Liver Injury Diseases 0.000 description 1
- 206010072268 Drug-induced liver injury Diseases 0.000 description 1
- 206010019799 Hepatitis viral Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- PQLVXDKIJBQVDF-UHFFFAOYSA-N acetic acid;hydrate Chemical compound O.CC(O)=O PQLVXDKIJBQVDF-UHFFFAOYSA-N 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960002246 beta-d-glucopyranose Drugs 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- DTGRQRFFKHYRJG-UHFFFAOYSA-N magnesium tetrahydrate Chemical compound O.O.O.O.[Mg+2] DTGRQRFFKHYRJG-UHFFFAOYSA-N 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 201000001862 viral hepatitis Diseases 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/60—Construction of the column
- G01N30/6052—Construction of the column body
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention provides a high performance liquid chromatography method for separating and detecting magnesium isoglycyrrhetate and related substances thereof and application thereof. Wherein the chromatographic column stationary phase is octadecylsilane chemically bonded silica, and the mobile phase is dipotassium hydrogen phosphate solution-acetonitrile, and the related substances are selected from any one of related substances 1, 2, 3,4, 5, 6, 7, 8 and 9 or a combination thereof. The method can realize the efficient separation and detection of magnesium isoglycyrrhetate and related substances thereof, and can effectively control the quality of medicines.
Description
Technical Field
The invention relates to the technical field of medicine analysis, in particular to a high performance liquid chromatography for separating and detecting magnesium isoglycyrrhetate and related substances thereof and application thereof.
Background
Magnesium isoglycyrrhetate is a hepatocyte protective agent, and has the effects of resisting inflammation, protecting liver cell membrane and improving liver function. Magnesium isoglycyrrhetate is clinically used for treating chronic viral hepatitis and acute drug-induced liver injury at present. The chemical name of the compound is (18 alpha, 20 beta) -20-carboxyl-11-oxo-30-nor olean-12-alkene-3 beta-yl-2-O-beta-D-glucopyranose aldehyde acid group-alpha-D-glucopyranose aldehyde acid magnesium tetrahydrate, and the chemical structure is as follows:
the presence of related substances is directly related to the quality and safety of the pharmaceutical product. The related substances comprise impurities brought by raw materials of the medicine, related substances generated in the process, related substances related to degradation and the like, which can directly influence the purity and quality of the medicine, even cause serious adverse reactions and influence the effectiveness and safety of clinical medicine. In order to improve the product quality of magnesium isoglycyrrhetate and ensure the safety and effectiveness of clinical medication, the related substance condition of the magnesium isoglycyrrhetate needs to be fully researched and an applicable separation detection method needs to be developed, impurities are controlled within the safety limit range, the controllable quality of the medicine is realized, and the safety and effectiveness of the clinical medication are ensured.
Disclosure of Invention
An object of one aspect of the present invention is to provide a high performance liquid chromatography for separating and detecting magnesium isoglycyrrhetate and related substances thereof in a magnesium isoglycyrrhetate bulk drug or preparation, wherein a chromatographic column stationary phase is octadecylsilane chemically bonded silica, a chromatographic column temperature is 20-40 ℃, a mobile phase is dipotassium hydrogen phosphate solution-acetonitrile, a mobile phase flow rate is 0.8ml/min-1.2ml/min, a sample injection volume is 10 μl-30 μl, the high performance liquid chromatography is provided with an ultraviolet detector, a detection wavelength of the ultraviolet detector is 240nm-260nm, and the related substances are selected from any one of related substances 1, 2, 3,4, 5, 6, 7, 8 and 9 or a combination thereof:
in a preferred embodiment of the present invention, the octadecylsilane chemically bonded silica column is a commercially available octadecylsilane chemically bonded silica column, preferably ThermoFisher, YMC, phenomenex, ES, merck, agilent, kromasil, agela, techmate, waters or any of the medium spectrum red company.
In a preferred embodiment of the present invention, the octadecylsilane chemically bonded silica column is selected from any one of Eclipse Plus C18, kromasil 100-5-C18, kromasil Eternity-5-C18, kromasil EternityXT-10-C18, kromasil 100-10-C18, kromasil 300-5-C18, YMC Pack ODS-AQ C18, YMC Pack ODS-AM C18, YMC Triart C18, phenomenex kinetex C C18, titank C18, ES-C18, epic C18, ZORBAX SB-C18, ZORBAX 300SB-C18, porosiwell 120EC-C18, XDB-C18, TC-C18, extend-C18, bonshell ASB 18, venusil HLP 18, venusil C18 Plus, venusil XBP 18 (A), innul XBP 18 (B), venusil C18, UG C18, UG 1, UG C18, or UG-phase C18, or combination thereof.
In a preferred embodiment of the present invention, the octadecylsilane chemically bonded silica column is preferably YMC Pack ODS-AQ C18.6X105 mm,3 μm.
In a preferred embodiment of the present invention, the column temperature of the chromatographic column is 25 ℃ to 35 ℃, preferably 28 ℃ to 32 ℃, more preferably 30 ℃.
In the preferred technical scheme of the invention, the volume ratio of the dipotassium hydrogen phosphate solution to acetonitrile in the mobile phase is 60-90:40-10, preferably 60-80:40-20, more preferably 65-75:35-25, more preferably 69-71:31-29.
In a preferred embodiment of the invention, the pH of the dipotassium hydrogen phosphate solution in the mobile phase is from 6.0 to 8.0, preferably from 6.5 to 7.5, more preferably from 6.8 to 7.2, and even more preferably 7.0.
In a preferred embodiment of the present invention, the dipotassium hydrogen phosphate solution in the mobile phase is pH-adjusted with a pH-adjusting agent selected from any one or a combination of formic acid, acetic acid, phosphoric acid and trifluoroacetic acid, preferably phosphoric acid.
In a preferred embodiment of the invention, the concentration of the dipotassium hydrogen phosphate solution in the mobile phase is 0.01M-0.1M, preferably 0.03M-0.08M, more preferably 0.048M-0.052M.
In a preferred embodiment of the invention, the dipotassium hydrogen phosphate solution in the mobile phase has a concentration of 0.05M and is adjusted to pH 7.0 with phosphoric acid.
In a preferred embodiment of the present invention, the flow rate of the mobile phase is 0.9ml/min to 1.1ml/min, preferably 1.0ml/min.
In a preferred embodiment of the present invention, the mobile phase is eluted isocratically or in a gradient, preferably isocratically.
In a preferred embodiment of the invention, the sample volume is 15. Mu.l-25. Mu.l, preferably 18. Mu.l-22. Mu.l.
In a preferred embodiment of the present invention, the detection wavelength of the ultraviolet detector is 245nm to 255nm, preferably 250nm.
In a preferred embodiment of the present invention, the peak-out sequence is related substance 1, related substance 2, related substance 3, related substance 4, related substance 5, magnesium isoglycyrrhetate, related substance 6, related substance 7, related substance 8, and related substance 9 in this order, wherein the degree of separation between the magnesium isoglycyrrhetate peak and the related substance 5 peak, between the related substance 6 peaks, and between the respective related substance peaks is greater than 1.5.
In a preferred embodiment of the present invention, the high performance liquid chromatography comprises the steps of:
1) Preparing a solution:
test solution: taking a proper amount of magnesium isoglycyrrhetate, dissolving the magnesium isoglycyrrhetate in a mobile phase, and diluting the magnesium isoglycyrrhetate into a solution containing about 1mg of magnesium isoglycyrrhetate in each 1 ml;
mixing solution: respectively taking proper amounts of magnesium isoglycyrrhetate, related substances 1, 2, 3,4, 5, 6, 7, 8 and 9, dissolving and diluting with mobile phase to obtain a mixed solution containing 1mg of magnesium isoglycyrrhetate and 1-9 μg of related substances 1-9;
2) Taking a chromatographic column YMC Pack ODS-AQ C18.6X250 mm and 3 mu M as a stationary phase, wherein the column temperature is 28-32 ℃, the mobile phase is dipotassium hydrogen phosphate solution-acetonitrile, the concentration of the dipotassium hydrogen phosphate solution is 0.048-0.052M, the pH value of the dipotassium hydrogen phosphate solution is adjusted to 6.8-7.2 by phosphoric acid, and the volume ratio of the dipotassium hydrogen phosphate solution to the acetonitrile is 69-71:31-29, injecting the eluted sample into a liquid chromatograph, wherein the sample injection amount is 20 μl; the detection wavelength of the ultraviolet detector is 250nm; the flow rate was 0.9ml-1.1ml per minute, and the chromatogram was eluted and recorded.
The invention also aims to provide the application of the high performance liquid chromatography for separating and detecting magnesium isoglycyrrhetate and related substances thereof in the high performance separation and detection of magnesium isoglycyrrhetate bulk drugs or preparations.
Unless otherwise indicated, when the invention relates to a percentage between liquids, the percentages are volume/volume percentages; the invention relates to the percentage between liquid and solid, said percentage being volume/weight percentage; the invention relates to the percentage between solids and liquids, the percentage being weight/volume percentage; the balance being weight/weight percent.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention scientifically screens and optimizes the elution condition of the high performance liquid chromatography, realizes effective separation and detection of 10 compounds which have approximate structures and are difficult to separate, such as magnesium isoglycyrrhetate and related substances thereof, has good separation effect, can effectively control the quality of medicines, reduces adverse reactions possibly occurring in clinical experiments and treatment of medicines, and ensures the effectiveness and safety of the medication of patients.
(2) The method has the advantages of short time consumption, time cost saving, economic benefit improvement and suitability for industrialized and large-scale production. The flow compatibility of the invention is easy to prepare, and the invention is prepared according to a conventional method.
(3) The method has strong specificity, all related substances and blank solvents do not interfere with each other, and the separation degree between the peaks of all the components is more than 1.5; the sensitivity of the chromatographic condition detection related substances is high; when the chromatographic conditions are slightly changed, the peak separation of each component is not affected, and the durability of the method is good.
Drawings
Fig. 1 is a high performance liquid chromatogram of example 1.
Fig. 2 is a high performance liquid chromatogram of example 2.
FIG. 3 is a high performance liquid chromatogram of example 3.
Fig. 4 is a high performance liquid chromatogram of example 4.
Fig. 5 is a high performance liquid chromatogram of example 5.
FIG. 6 is a high performance liquid chromatogram of example 6.
FIG. 7 is a high performance liquid chromatogram of example 7.
FIG. 8 is a high performance liquid chromatogram of example 8.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail hereinafter with reference to the accompanying drawings. It should be noted that, without conflict, the embodiments of the present invention and features in the embodiments may be arbitrarily combined with each other.
EXAMPLE 1 HPLC separation detection of magnesium isoglycyrrhetate and related substances
Liquid chromatograph: shimadzu corporation LC-20A
Chromatographic column: GL Inertsil ODS-3, 4.6mm.times.250 mm,5 μm
Column temperature: 30 DEG C
Mobile phase: acetonitrile-glacial acetic acid-water (38:1:61)
Flow rate: 1ml/min
Ultraviolet detector detection wavelength: 250nm
Sample injection volume: 20 μl of
Mixing solution: magnesium isoglycyrrhetate, related substance 1, related substance 2, related substance 3, related substance 4, related substance 5, related substance 6, related substance 7, related substance 8 and related substance 9 are dissolved in a mobile phase and diluted to prepare a mixed solution containing about 1mg of magnesium isoglycyrrhetate and about 1-10 μg of each of related substances 1-9 in each 1 ml.
The chromatographic results of the mixed solution are shown in FIG. 1.
The results show that: multiple compounds do not show peaks, and effective separation cannot be achieved, and product quality cannot be controlled.
EXAMPLE 2 HPLC separation detection of magnesium isoglycyrrhetate and related substances
Liquid chromatograph: shimadzu corporation LC-20A
Chromatographic column: waters Xbridge C18.6X250 mm,5 μm
Column temperature: 30 DEG C
Mobile phase: 0.1M dipotassium hydrogen phosphate solution (pH adjusted to 7.0 with phosphoric acid) -acetonitrile (80:20)
Flow rate: 1ml/min
Ultraviolet detector detection wavelength: 250nm
Sample injection volume: 20 μl of
Mixing solution: magnesium isoglycyrrhetate, related substance 1, related substance 2, related substance 3, related substance 4, related substance 5, related substance 6, related substance 7, related substance 8 and related substance 9 are dissolved in a mobile phase and diluted to prepare a mixed solution containing about 1mg of magnesium isoglycyrrhetate and about 1-10 μg of each of related substances 1-9 in each 1 ml.
The chromatographic results of the mixed solution are shown in FIG. 2.
The results show that: multiple compounds do not show peaks, and effective separation cannot be achieved, and product quality cannot be controlled.
EXAMPLE 3 HPLC separation detection of magnesium isoglycyrrhetate and related substances
Liquid chromatograph: shimadzu corporation LC-20A
Chromatographic column: agilent ZOBAX Eclipse C18 4.6X250 mm,5 μm
Column temperature: 30 DEG C
Mobile phase: 0.1M dipotassium hydrogen phosphate solution (pH adjusted to 7.0 with phosphoric acid) -acetonitrile (80:20)
Flow rate: 1ml/min
Ultraviolet detector detection wavelength: 250nm
Sample injection volume: 20 μl of
Mixing solution: magnesium isoglycyrrhetate, related substance 1, related substance 2, related substance 3, related substance 4, related substance 5, related substance 6, related substance 7, related substance 8 and related substance 9 are dissolved in a mobile phase and diluted to prepare a mixed solution containing about 1mg of magnesium isoglycyrrhetate and about 1-10 μg of each of related substances 1-9 in each 1 ml.
The chromatographic results of the mixed solution are shown in FIG. 3.
The results show that: multiple compounds do not show peaks, and effective separation cannot be achieved, and product quality cannot be controlled.
EXAMPLE 4 HPLC separation detection of magnesium isoglycyrrhetate and related substances
Liquid chromatograph: shimadzu corporation LC-20A
Chromatographic column: YMC Pack ODS-AQ 4.6X105 mm,3 μm
Column temperature: 30 DEG C
Mobile phase: 0.1M dipotassium hydrogen phosphate solution (pH adjusted to 7.0 with phosphoric acid) -acetonitrile (80:20)
Flow rate: 1ml/min
Ultraviolet detector detection wavelength: 250nm
Sample injection volume: 20 μl of
Mixing solution: magnesium isoglycyrrhetate, related substance 1, related substance 2, related substance 3, related substance 4, related substance 5, related substance 6, related substance 7, related substance 8 and related substance 9 are dissolved in a mobile phase and diluted to prepare a mixed solution containing about 1mg of magnesium isoglycyrrhetate and about 1-10 μg of each of related substances 1-9 in each 1 ml.
The chromatographic results of the mixed solution are shown in FIG. 4.
The results show that: the separation of the compounds is improved, but effective separation can not be realized, and the product quality can not be controlled.
EXAMPLE 5 HPLC separation detection of magnesium isoglycyrrhetate and related substances
Liquid chromatograph: shimadzu corporation LC-20A
Chromatographic column: YMC Pack ODS-AQ 4.6X105 mm,3 μm
Column temperature: 30 DEG C
Mobile phase: 0.05M dipotassium hydrogen phosphate solution (pH adjusted to 7.0 with phosphoric acid) -acetonitrile (90:10)
Flow rate: 1ml/min
Ultraviolet detector detection wavelength: 250nm
Sample injection volume: 20 μl of
Mixing solution: magnesium isoglycyrrhetate, related substance 1, related substance 2, related substance 3, related substance 4, related substance 5, related substance 6, related substance 7, related substance 8 and related substance 9 are dissolved in a mobile phase and diluted to prepare a mixed solution containing about 1mg of magnesium isoglycyrrhetate and about 1-10 μg of each of related substances 1-9 in each 1 ml.
The chromatographic results of the mixed solution are shown in FIG. 5.
The results show that: the separation is improved, but the impurities are not peaked, and effective separation cannot be realized.
EXAMPLE 6 HPLC separation detection of magnesium isoglycyrrhetate and related substances
Liquid chromatograph: shimadzu corporation LC-20A
Chromatographic column: YMC Pack ODS-AQ 4.6X105 mm,3 μm
Column temperature: 30 DEG C
Mobile phase: 0.05M dipotassium hydrogen phosphate solution (pH adjusted to 7.0 with phosphoric acid) -acetonitrile (60:40)
Flow rate: 1ml/min
Ultraviolet detector detection wavelength: 250nm
Sample injection volume: 20 μl of
Mixing solution: magnesium isoglycyrrhetate, related substance 1, related substance 2, related substance 3, related substance 4, related substance 5, related substance 6, related substance 7, related substance 8 and related substance 9 are dissolved in a mobile phase and diluted to prepare a mixed solution containing about 1mg of magnesium isoglycyrrhetate and about 1-10 μg of each of related substances 1-9 in each 1 ml.
The chromatographic results of the mixed solution are shown in FIG. 6.
The results show that: the system cannot realize the effective separation of the main component and a plurality of impurity compounds, and cannot control the quality of products.
EXAMPLE 7 HPLC separation detection of magnesium isoglycyrrhetate and related substances
Liquid chromatograph: shimadzu corporation LC-20A
Chromatographic column: YMC Pack ODS-AQ 4.6X105 mm,3 μm
Column temperature: 30 DEG C
Mobile phase: 0.05M dipotassium hydrogen phosphate solution (pH adjusted to 7.0 with phosphoric acid) -acetonitrile (70:30)
Flow rate: 1ml/min
Ultraviolet detector detection wavelength: 250nm
Sample volume 20. Mu.l
Mixing solution: magnesium isoglycyrrhetate, related substance 1, related substance 2, related substance 3, related substance 4, related substance 5, related substance 6, related substance 7, related substance 8 and related substance 9 are dissolved in a mobile phase and diluted to prepare a mixed solution containing about 1mg of magnesium isoglycyrrhetate and about 1-10 μg of each of related substances 1-9 in each 1 ml.
The chromatographic results of the mixed solution are shown in FIG. 7.
The results show that: the magnesium isoglycyrrhetate can be well separated from adjacent impurities and impurity compounds.
EXAMPLE 8 specificity of the HPLC separation detection method of the invention
Liquid chromatograph: shimadzu corporation LC-20A
Chromatographic column: YMC Pack ODS-AQ 4.6X105 mm,3 μm
Column temperature: 30 DEG C
Mobile phase: 0.05M dipotassium hydrogen phosphate solution (pH adjusted to 7.0 with phosphoric acid) -acetonitrile (70:30)
Flow rate: 1ml/min
Ultraviolet detector detection wavelength: 250nm
Sample volume 20. Mu.l
Solvent: 0.05M dipotassium hydrogen phosphate solution (pH adjusted to 7.0 with phosphoric acid) -acetonitrile (70:30);
positioning solution: respectively dissolving magnesium isoglycyrrhetate, related substances 1, 2, 3,4, 5, 6, 7, 8 and 9 in solvent, and diluting to obtain solution containing about 10-100 μg per 1 ml;
mixing solution: respectively taking proper amounts of magnesium isoglycyrrhetate, related substances 1, 2, 3,4, 5, 6, 7, 8 and 9, dissolving with solvent, and diluting to obtain a mixed solution containing 1mg of magnesium isoglycyrrhetate and 1-9 μg of related substances 1-9;
the chromatographic results of the mixed solution are shown in Table 1 below and FIG. 8.
TABLE 1
The results show that: the separation degree between each impurity peak and between the main component and the adjacent impurity peak is more than 1.5, and the method has good specificity.
Example 9 sensitivity of HPLC separation detection method of the present invention
Chromatographic conditions were the same as in example 8.
The preparation method of the solution comprises the following steps: magnesium isoglycyrrhetate, related substance 1, related substance 2, related substance 3, related substance 4, related substance 5, related substance 6, related substance 7, related substance 8 and related substance 9 are respectively taken, a mixed solution of about 0.5 mug of magnesium isoglycyrrhetate and each of the related substances 1-9 in each 1ml is prepared by stepwise dilution with a solvent, the mixed solution is taken as a quantitative limiting solution (the signal to noise ratio of each component main peak is required to be not less than 10), and the quantitative limiting solution is taken as a detection limiting solution (the signal to noise ratio of each component main peak is required to be not less than 3) by diluting 3 times with the solvent.
The limit of detection and the limit of quantification result are shown in tables 2 and 3, respectively, below.
TABLE 2
TABLE 3 Table 3
The results show that: the method for detecting the magnesium isoglycyrrhetate and the related substances has high sensitivity, and the quantitative detection can be finished when the compounds are low to ng level.
EXAMPLE 10 durability of the HPLC separation detection method of the present invention
Chromatographic conditions were the same as in example 8.
Solvent: 0.05M dipotassium hydrogen phosphate solution (pH adjusted to 7.0 with phosphoric acid) -acetonitrile (70:30);
mixing solution: magnesium isoglycyrrhetate, related substance 1, related substance 2, related substance 3, related substance 4, related substance 5, related substance 6, related substance 7, related substance 8, and related substance 9 are dissolved in a solvent and diluted to prepare a mixed solution containing about 1mg of magnesium isoglycyrrhetate and about 1-10 μg of each of related substances 1-9 per 1 ml.
The results of the degree of separation are shown in Table 4 below.
TABLE 4 Table 4
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The results show that: after the chromatographic conditions in the embodiment 8 of the invention are partially adjusted, the separation degree of each component still meets the requirements, and the method has good durability.
Although the embodiments disclosed in the present invention are described above, the embodiments are merely used to facilitate understanding of the present invention, and are not intended to limit the present invention. Any person skilled in the art can make any modification and variation in form and detail without departing from the spirit and scope of the present disclosure, but the scope of the present disclosure is to be determined by the appended claims.
Claims (10)
1. A high performance liquid chromatography for separating and detecting magnesium isoglycyrrhetate and related substances in magnesium isoglycyrrhetate bulk drug or preparation, wherein a chromatographic column stationary phase is octadecylsilane chemically bonded silica, the chromatographic column temperature is 20-40 ℃, a mobile phase is dipotassium hydrogen phosphate solution-acetonitrile, the flow rate of the mobile phase is 0.8ml/min-1.2ml/min, the sample injection volume is 10 μl-30 μl, the high performance liquid chromatography is provided with an ultraviolet detector, the detection wavelength of the ultraviolet detector is 240nm-260nm, and the related substances are selected from any one or combination of related substances 1, 2, 3,4, 5, 6, 7, 8 and 9:
2. the high performance liquid chromatography according to claim 1, wherein the octadecylsilane chemically bonded silica column is a commercially available octadecylsilane chemically bonded silica column, preferably ThermoFisher, YMC, phenomenex, ES, merck, agilent, kromasil, agela, techmate, waters or any of the medium spectrum red companies.
3. The high performance liquid chromatography of any one of claims 1-2, wherein the octadecylsilane chemically bonded silica column is selected from Eclipse Plus C18, kromasil 100-5-C18, kromasil Eternity-5-C18, kromasil EternityXT-10-C18, kromasil 100-10-C18, kromasil 300-5-C18, YMC Pack ODS-AQ C18, YMC Pack ODS-AM C18, YMC branch C18, phenomenex kinetex C, titank C18, ES-C18, epic C18, ZORBAX SB-C18, ZORBAX 300SB-C18, porose 120EC-C18, XDB-C18, TC-C18, extented-C18, bonchel ASB 18, venuquel HLP 18, venusil C18 Plus, venup 18B (a), venuqp 18B), venuq C18, or any combination thereof.
4. The method according to claim 3, wherein the octadecylsilane chemically bonded silica column is YMC Pack ODS-AQ C18.6X105 mm,3 μm.
5. The high performance liquid chromatography according to any one of claims 1 to 4, wherein the column temperature of the chromatography column is 25 ℃ to 35 ℃, preferably 28 ℃ to 32 ℃, more preferably 30 ℃, and the volume ratio of dipotassium hydrogen phosphate solution to acetonitrile in the mobile phase is 60 to 90:40-10, preferably 60-80:40-20, more preferably 65-75:35-25, more preferably 69-71:31-29, the pH of the dipotassium hydrogen phosphate solution in the mobile phase is 6.0-8.0, preferably 6.5-7.5, more preferably 6.8-7.2, more preferably 7.0, the pH of the dipotassium hydrogen phosphate solution in the mobile phase is adjusted with a pH adjuster selected from any one or combination of formic acid, acetic acid, phosphoric acid and trifluoroacetic acid, preferably phosphoric acid, and the concentration of the dipotassium hydrogen phosphate solution in the mobile phase is 0.01-0.1M, preferably 0.03-0.08M, more preferably 0.048-0.052M.
6. The high performance liquid chromatography according to any one of claims 1 to 5, wherein the concentration of the dipotassium hydrogen phosphate solution in the mobile phase is 0.05M, and wherein the pH is adjusted to 7.0 with phosphoric acid.
7. The high performance liquid chromatography according to any one of claims 1 to 6, wherein the flow rate of the mobile phase is 0.9ml/min to 1.1ml/min, preferably 1.0ml/min, the mobile phase is isocratic or gradient elution, preferably isocratic, the sample volume is 15 μl to 25 μl, preferably 18 μl to 22 μl, and the detection wavelength of the ultraviolet detector is 245nm to 255nm, preferably 250nm.
8. The method according to any one of claims 1 to 7, wherein the peak order is in the order of related substance 1, related substance 2, related substance 3, related substance 4, related substance 5, magnesium isoglycyrrhetate, related substance 6, related substance 7, related substance 8, and related substance 9, and wherein the degree of separation between the magnesium isoglycyrrhetate peak and the related substance 5 peak, the related substance 6 peak, and between each related substance peak is greater than 1.5.
9. The high performance liquid chromatography according to any one of claims 1-8, wherein the high performance liquid chromatography comprises the steps of:
1) Preparing a solution:
test solution: taking a proper amount of magnesium isoglycyrrhetate, dissolving the magnesium isoglycyrrhetate in a mobile phase, and diluting the magnesium isoglycyrrhetate into a solution containing about 1mg of magnesium isoglycyrrhetate in each 1 ml;
mixing solution: respectively taking proper amounts of magnesium isoglycyrrhetate, related substances 1, 2, 3,4, 5, 6, 7, 8 and 9, dissolving with solvent, and diluting to obtain a mixed solution containing 1mg of magnesium isoglycyrrhetate and 1-9 μg of related substances 1-9;
2) The chromatographic column YMC Pack ODS-AQ C18.6X250 mm and 3 μm is used as stationary phase, the column temperature is 28-32 ℃,
the mobile phase is dipotassium hydrogen phosphate solution-acetonitrile, the concentration of the dipotassium hydrogen phosphate solution is 0.048M-0.052M, the pH value of the dipotassium hydrogen phosphate solution is adjusted to 6.8-7.2 by phosphoric acid, and the volume ratio of the dipotassium hydrogen phosphate solution to the acetonitrile is 69-71:31-29, injecting the eluted sample into a liquid chromatograph, wherein the sample injection amount is 20 μl; the detection wavelength of the ultraviolet detector is 250nm; the flow rate was 0.9ml-1.1ml per minute, and the chromatogram was eluted and recorded.
10. The use of high performance liquid chromatography according to any one of claims 1-9 for high performance separation and detection of magnesium isoglycyrrhetate and related substances in magnesium isoglycyrrhetate bulk drugs or formulations.
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